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Application of Phage Chip as Protein Chip or Gene Chip

A phage and chip technology, applied in the field of biomedicine, achieves particle stability, facilitates optimization of preparation conditions and standardized operations, control of chip quality, and reduces processing difficulty

Inactive Publication Date: 2012-02-01
XI AN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the random peptide sequences displayed by the phage random peptide library have no similarity with natural molecules, they still have similar functions or antigenicity to natural molecules

Method used

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  • Application of Phage Chip as Protein Chip or Gene Chip

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0016] 2. Preparation of phage chip:

[0017] Using artificial or non-artificial methods and corresponding equipment, the phages displaying specific foreign peptides or antigenic epitopes obtained above are coupled to different chip substrates according to the designed array pattern, such as glass slides, silicon chips, ceramic chips, etc. Phage chips can be obtained by using different microbeads such as sheets, nylon membranes, PVDF membranes, NC membranes, polyacrylamide gels, magnetic beads, and agarose. At the same time, the above-mentioned chip base may or may not undergo different activation treatments such as aldehydes, thiols, aminations, or epoxy groups, so as to facilitate the display of phages on the surface of the glass slide on the chip base in various possible ways.

[0018] 3. Application of phage chip:

[0019] 3.1 The phage chip provided by the present invention can be used as a protein chip displaying foreign peptides or antigenic epitopes for use in antibod...

Embodiment 1

[0036] Embodiment 1: Preparation of phage chip

[0037] 1. Screening and identification of phages displaying foreign peptides or antigenic epitopes

[0038] Method 1: Screening and Identification of Phage Random Peptide Libraries

[0039] (1) Collect and separate serum, antibodies, body fluids or tissue extracts from human or experimental animals by conventional methods.

[0040](2) "Biopanning" method to screen phage random peptide library: take the amplified and diluted phage peptide library (1.0×10 11 pfu), and 300ng of normal rabbit IgG solution into 2ml of binding solution, 37°C, shake and incubate at 50rpm, 1h for pre-adsorption, add protein G-labeled magnetic beads 50μl, 37°C, shake and incubate at 50rpm for 20min, magnetic adsorption for 2min, take Precipitate with 1 / 6 volume of 2.5M PEG / NaCl, dissolve in 200μl TBS, add to 2ml of human or experimental animal serum, antibody, body fluid or tissue extraction solution containing 300ng antibody, incubate at 37°C for 1h, ...

Embodiment 2

[0048] Example 2: Application of phage chip in antibody detection

[0049] Method 1: Block the prepared phage chip with 5% skimmed milk at room temperature for 2 hours, add 1:500 diluted sample to be tested, incubate at room temperature for 2 hours, wash with TBST (TBS containing 0.5% Tween-20), 10min×3, 1: 1000 diluted HRP-labeled goat anti-rabbit IgG was incubated at room temperature for 20 minutes, washed with TBST for 10 minutes×3, and developed with DAB. The specific antibody in the sample can be detected ( figure 1 ).

[0050] Method 2: The antibody in the sample was separated by a conventional method, and the antibody was labeled by the Cy3 fluorescent labeling method (the Cy3 fluorescent labeling kit was purchased from Pharmacia). Block the phage chip with 5% skimmed milk at room temperature, add Cy3-labeled sample to be detected, incubate at room temperature for 2 hours, wash with TBST (TBS containing 0.5% Tween-20), 10min×3, and interpret the results with a fluores...

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Abstract

The invention discloses the application of a phage chip as a protein chip or a gene chip. The present invention obtains phage clones displaying specific exogenous peptides (or antigenic epitopes) by screening phage peptide libraries or constructing phage display libraries, and displays different exogenous peptides in an automatic or non-automatic manner according to a pre-designed array pattern. The phages were directly coupled to the same chip substrate to obtain a phage chip. The chip can be used not only as a protein chip displaying exogenous peptides, but also for the detection, screening and identification of antibodies, ligands and specific binders in natural or synthetic compounds in various samples of human and experimental animals, and as a clone The gene chip of related genes is used for the detection of related genes. The phage chip has the remarkable characteristics of low cost, simple preparation, high stability, high specificity, easy long-term storage and the like.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to the preparation and application of a phage chip, in particular to the preparation and application of a phage biochip displaying exogenous antigen peptides. In the present invention, different phages displaying exogenous peptides are fixed on the chip base according to the designed array method to obtain a phage chip, which can be used as a protein chip displaying exogenous peptides for various samples of human body or experimental animals The detection, identification and screening of antibodies, ligands and specific binders in natural or synthetic compounds can also be used as a gene chip for the detection, identification and screening of specific genes. Background technique [0002] Biochip mainly refers to the construction of a miniature biochemical analysis system on the surface of a solid chip through micromachining and microelectronics technology, so as to realize accurate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 杨军张明娟李宗芳苏宝山
Owner XI AN JIAOTONG UNIV
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